High-Resolution Imaging of Single Fluorescent Molecules with the Optical Near-Field of a Metal Tip H. G. Frey, S. Witt, K. Felderer, and R. Guckenberger, Phys. Rev. Lett. 93, (2004). Marc McGuigan Journal Club Monday, April 10, 2006

Outline Introduction  Near Field Microscopy  Purpose Experimental Setup Sample Preparation Results Data Model Conclusion

Beating the Diffraction Limit Alternatives Scanning Tunneling Microscope Atomic Force Microscope Scanning Electron Microscope Transmission Electron Microscope Why use visible light? Contrast Easier Sample Preparation

History 1928 – Synge Idea (1)  Strong light source behind thin metal film  100 nm diameter hole to illuminate biological sample  Sample less than 100 nm away from source  Discusses ideas in letters to Albert Einstein (2) 1972 – E. A. Ash and G. Nicholls (3)  Passed microwaves (3 cm) through 1.5 mm aperture  Scanned over grating and were able to resolve 0.5 mm lines and 0.5 mm gaps in grating 1984  Pohl, Denk, Duerig (IBM) (SNOM)  Lewis group (Cornell) (NSOM)  Subwavelength aperture at apex of sharp transparent probe tip that is coated with metal Diagram Source: Molecular Expressions Optical Microscopy Primer,

Evanescent Waves Diagram Source: K. Iizuka, Elements of Photonics: In Free Space and Special Media, Volume 1 (John Wiley & Sons, New York, 2002). (4) Total Internal ReflectionWave vectors propagating in k space

Evanescent Waves To satisfy boundary conditions: This can be re-written as: The value of k xn is imaginary for high values of m and the waves are evanescent waves Diagram Source: K. Iizuka, Elements of Photonics: In Free Space and Special Media, Volume 1 (John Wiley & Sons, New York, 2002). Above d c k x is always imaginary and all the waves in x are evanescent waves. Evanescent Waves on a Corrugated Metal Surface Evanescent Waves on an Array of Metal Pins

Modes of Near Field Imaging (a) Aperture NSOM(b)Aperture- less NSOM (c)Scanning tunneling optical microscope Different types of scanning near field optical microscopes NSOM Configurations (a) collection (b) illumination (c) collection/illumination (d) oblique collection(e) oblique illumination (f) Dark field Diagram (left) Source: M. A. Paesler and P. J. Moyer, Near Field Optics: Theory, Instrumentation, and Applications (John Wiley & Sons, New York, 1996). Diagram (right) Source: B. Hecht et al., J. Chem. Phys. 112, 7761 (2000).

NSOM Setup Standard NSOM Setup (a) Illumination (b)Collection and Redistribution (c) Detection Tips (5) Heating and pulling method - Optical fiber is heated with CO 2 laser and pulled on both sides of heated area Chemical etching method - Hydrofluoric acid used to etch glass fiber Fiber coated with metal Nanoparticle (Tip Enhanced) Diagram (left) Source: B. Hecht et al., J. Chem. Phys. 112, 7761 (2000). Diagram (right) Source: Molecular Expressions Optical Microscopy Primer,

Aperture NSOM Resolution: nm Problems (6) Difficult to create smooth aluminum coating on nanometer scale Flat ends of the probes are not good for high resolution topographic imaging Absorption of light by metal coating causes significant heating Aluminum-coated aperture probes (a), (b) prepared by pulling (c), (d) prepared by etching 300 nm (a), (c) macroscopic shape, SEM and optical image (b), (d) SEM close-up of the aperture region Diagram (left) Source: B. Hecht et al., J. Chem. Phys. 112, 7761 (2000). Diagram (right) Source: Molecular Expressions Optical Microscopy Primer,

Tip-Enhanced NSOM Resolution: nm Causes for Enhanced Electric Field: (7) Electrostatic lightning rod effect (depends on geometry) Surface plasmon resonances (depend on excitation wavelength and geometry) Induced surface charge density in metal probe Left: Incident wave polarized perpendicular to tip axis Right: Incident wave polarized along tip axis Need large near field enhancement so the signal can be detected in the far field The incident field should be polarized along the tip axis to maximize field enhancement Schematic of experimental setup for tip-enhanced near field Diagram (left) Source: A. Hartschuh, M. R. Beversluis, A. Bouhelier, and L. Novotny, Phil. Trans. R. Soc. Lond. A. 362, 807 (2004). (7) Diagram (right) Source: L. Novotny, R. X. Bian, and X. S. Xie, Phys. Rev. Lett. 79, 645 (1997). (8)

NSOM and Fluorescence Aperture NSOM resolution ~ 50 nm Tip-enhanced better resolution high background signal Bleaching of dyes One solution: two-photon excitation Two-photon excitation is a nonlinear process Detected signal is proportional to the square of the intensity enhancement factor (6) Illuminated area of sample : S = 10 5 nm 2 Intensity enhanced area under tip : σ = 100 nm 2 Simultaneous topographic image (a) and near-field two- photon excited fluorescence image (b) of J-aggregates of PIC dye in PVS film on a glass substrate. Diagram (left) Source: E. J. Sanchez, L. Novotny, and X. S. Xie, Phys. Rev. Lett. 82, 4014 (1999).

Purpose Interest: “investigation of self-fluorescing or fluorescence labeled macromolecules at the single molecule level.” Challenge: combine optical and topographical resolution of NSOM with fluorophore sensitivity Results:  “highly resolved optical imaging of single dyes”  “high-resolution topographs”

“tip-on-aperture” probe Thin optical fiber in etching solution (10) Tip covered with Cr (for adhesion) then 200 nm Au for contrast in SEM Focus electron beam of SEM on the center of the aperture Electron-beam-deposited tip (EBD) formed (7 s, 8 kV) 3.5 nm Cr and 33 nm Al deposited by evaporation at 45 o Drawing of “tip-on-aperture probe with the DNA sample. SEM images of a “tip-on-aperture” probe (a)Before metallization Diagram (left) Source: H. G. Frey, F. Keilmann, A. Kriele, and R. Guckenberger, Appl. Phys. Lett. 81, 5030 (2002). (b) After metallization

Experimental Setup Light Source: argon laser (514 nm) Light coupled to glass fiber onto the sample Light transmitted through the sample and collected by objective (0.95 NA) on inverse light microscope Light filtered by 550 nm long pass filter Signal detected by APD Sample scanned ~ 1 micron per second Scanned at constant distance with shear force feedback Polarization of incident laser light adjusted to optimize S/N 1/3 of probes provide good fluorescence results

Sample Preparation DNA  Cy-3 fluorophores covalently bound to the termini of DNA  Samples prepared in a polymerase chain reaction (PCR) Mica Sheets  20 μl of 400 mM NiCl 2 solution in water  2 min later – solution bottled off and 30 μl drop of DNA (with Cy-3 label) solution applied to the sheet  10 min later – washed in ultrapure water and dried with nitrogen

Results 200 nm 25 nm Fitted tip radius: 12 nm Fluorescence image of single Cy-3 dye molecules, which appear mostly as double maxima. FWHM = 10 nm Zoomed image of a dye molecule together with a section along the line (three lines average). Enlarged image of a bleaching event from one scan line (oriented vertically) to the next one. 25 nm

Data Model Dye molecule excitation proportional to squared field component parallel to dipole moment They believe that the field from the aperture light does not substantially influence the experiment Dye dipoles oriented vertically experience maximum excitation directly below the tip Inclined dyes display asymmetric peaks Vertical dye under the tip displays a circular structure Dye oriented in sample plane displays two symmetric maxima

Data Model Software: MATHLAB 6.5 (Mathworks) Classical Mirror Image Calculation Neglected:  Retroaction of dye dipole on tip dipole  Retardation effects Emission in direction of objective used to calculate final signal Lifetime without mirrorQuantum yield Index of refraction of medium with dipole Fit Parameters X, Y position of dye 3D orientation of dye Normalization factor for dye brightness and local background Parameters assumed constant Tip radius Tip-sample distance Quantum efficiency = 0.3

Results with Data Model Measurements Patterns calculated with parameters fitted to the measurements Tilt angle: 0 o Tilt angle: 68 o Tilt angle: 49 o Tilt angle: 20 o Tilt angle: 14 o Image size: 117 nm Fitted tip radius: 22nm Tip-dye distance (calculated): 1 nm Tip-dye distance (approach curve): 2-3 nm Why the discrepancy? Treatment of quenching effects neglects contributions with a stronger dependence on distance becoming important within 5 nm Tip apex flatter than a sphere Moon-like and ring-like patterns due to strong quenching effects when tip-dye distance below 3 nm As tip-dye distance increases central minimum decreases in size Total number of photon counts per pattern decreased by factor of 2 when tip- dye distance increases by 5 nm Fluorescence patterns of differently tilted dye molecules.

Results Fitted tip radius: 12 nm Analyzed dye molecules Note: A fitted parabola has been subtracted from each scan line to flatten the data (a) Topography together with calculated positions of analyzed dye molecules (c)Positions of dye molecules in (a) with tilt angles (upper number) and azimuth angles (lower numbers) from first approximation fits in (d) (b)Fluorescence image 200 nm (d)First approximation fits Green: Good fits Yellow: Problematic fits DNA with Cy-3 labeled termini on mica and corresponding data modeling. Accuracy in dye positions: 0.5 nm standard deviation Azimuth angle accurate to 5 o Accuracy of tilt angle better than 10 o

Results Fluorescence pattern of two dyes located close to each other 300 nm (a)Experimental data (b)Best fit when assuming two dyes for the encircled pattern (c)Difference between data (a) and fit (b) Single dye molecule

Conclusion Results  Near-field optical image of single fluorescent dye molecules at high resolution  High resolution topographic image of dye molecules Improvements  Optimize tip-aperture geometry to allow plasmon resonance Vary tip length Change material  Sharpen the metal tip to improve resolution

References 1. B. Hecht et al., J. Chem. Phys. 112, 7761 (2000). 2. M. A. Paesler and P. J. Moyer, Near Field Optics: Theory, Instrumentation, and Applications (John Wiley & Sons, New York, 1996). 3. Molecular Expressions Optical Microscopy Primer, K. Iizuka, Elements of Photonics: In Free Space and Special Media, Volume 1 (John Wiley & Sons, New York, 2002). 5. P. N. Prasad, Nanophotonics (John Wiley & Sons, Hoboken, 2004). 6. E. J. Sanchez, L. Novotny, and X. S. Xie, Phys. Rev. Lett. 82, 4014 (1999). 7. A. Hartschuh, M. R. Beversluis, A. Bouhelier, and L. Novotny, Phil. Trans. R. Soc. Lond. A. 362, 807 (2004). 8. L. Novotny, R. X. Bian, and X. S. Xie, Phys. Rev. Lett. 79, 645 (1997). 9. N. Anderson, A. Bouhelier, L. Novotny, J. Opt. A. 8, S227 (2006). 10. H. G. Frey, F. Keilmann, A. Kriele, and R. Guckenberger, Appl. Phys. Lett. 81, 5030 (2002).